Wolters Kluwer Health
may email you for journal alerts and information, but is committed
to maintaining your privacy and will not share your personal information without
your express consent. For more information, please refer to our Privacy Policy.

INTRODUCTION

The use of antiretroviral therapy has turned out HIV infection into a chronic manageable disease. Continuous lifelong therapy is, however, required to maintain virologic suppression, and a number of randomized trials have explored alternatives strategies of either structured1-4 or CD4-guided5-11 intermittent therapy (IT) to alleviate the constraints of daily uninterrupted therapy. Discontinuation of antiretroviral therapy was associated with an unacceptable increased risk of morbidity (both HIV- and non-HIV related) and mortality, which became significant after a few months, and these strategies are not recommended. Some patients are still stopping their therapy for short periods of time anyway, because of adherence problems or drug-related adverse events. But even a short interruption of antiretroviral therapy might lead to undesirable effects such as the emergence of resistance mutations or a drop in CD4 T-cell counts.12 Thrombocytopenia has also been reported as a potential consequence of interrupting antiretroviral therapy in a few patients, occurring weeks after treatment discontinuation, but its incidence and risk factors have not been fully investigated in recent trials.1,10,13 Furthermore, thrombocytopenia could be severe enough to lead to clinical manifestations.10 We took opportunity of the randomized Window ANRS-106 trial to assess the incidence and risk factors for thrombocytopenia among well-controlled HIV-infected patients with high CD4 cell counts and suppressed viral replication under antiretroviral therapy, who started a structured treatment interruption strategy.2 We also looked at the correlation between platelet counts, CD4 and CD8 T-cells counts, and plasma HIV RNA levels among patients interrupting their antiretroviral regimen.

METHODS

Patients

Between December 2001 and June 2003, 403 adult patients from 39 ANRS centers throughout France were enrolled in the ANRS 106 Window trial, a 96-week noninferiority, open-label trial. As previously reported, the objective of the trial was to assess the efficacy and safety of an intermittent antiretroviral therapy in patients with controlled HIV infection. Patients well tolerating combination antiretroviral therapy, with a nadir pretreatment CD4-cell count of 100/mm3 or more, a CD4-cell count above 450/mm3 at screening and plasma HIV-1 RNA levels below 400 copies/mL for at least the previous 6 months, were randomized to either continue their antiretroviral regimen (CT arm) or to switch to an 8-week off-8-week on structured IT (IT arm). Randomization was stratified on the use of efavirenz (nevirapine was not allowed in this trial), the CD4 cell count at entry (below or above 600 cells) and the nadir CD4 count (above or below 200 cells). Patients were assessed at baseline, then every 8 weeks until they completed the 96 weeks of follow-up. At each visit clinical data and blood specimens were collected. Platelet counts and CD4, CD8 T-cell counts, and plasma HIV RNA levels were measured on site. The primary endpoint was the proportion of patients reaching a confirmed CD4 cell count of less than 300/mm3.

Over 96 weeks, the proportion of patients meeting this endpoint was noninferior in the IT arm as compared with the CT arm [3.6% versus 1.5%, respectively, with an upper bound of the 95% confidence interval (CI) of the difference at 5.6%]. No AIDS-defining event was recorded, but 2 deaths occurred both in the IT arm. Also, more patients in the IT arm (14%) than in the CT arm (7%) experienced HIV-related clinical events (P = 0.04).

Study Objectives and Statistical Analysis

The first objective of this substudy was to estimate the incidence of thrombocytopenia during the study. Only the first occurrence of thrombocytopenia, defined as a platelet count below 150 × 103/mm3, was considered. The primary analysis was an intent-to-treat analysis in which data of all randomized patients who started the assigned treatment strategy were considered, whether this strategy was prematurely discontinued. A second on treatment analysis was also performed in which observed data from patients who remained under their assigned treatment strategy were considered for analysis. Discontinuation of the treatment strategy was defined as the discontinuation of antiretroviral therapy for 6 weeks or more in the CT arm, and by the use of antiretroviral therapy for 6 weeks or more during a period off treatment in the IT arm. Kaplan-Meir plots were performed and the Log-rank test was used to compare the time to the occurrence of thrombocytopenia between groups.

The second objective of the study was to identify baseline predictors of thrombocytopenia (platelets below <150 × 103/mm3). An univariate logistic regression model was used, including the following variables: age (by steps of 10 years), sex, history of AIDS-defining illness, nadir CD4 count (by steps of 50 cells), baseline CD4 T-cell counts (by steps of 100 cells), coinfection with hepatitis B or C, use of zidovudine or didanosine (ddI) in the baseline regimen (according to their efficacy in the treatment of HIV-related thrombocytopenia), use of nonnucleoside reverse transcriptase inhibitor in the baseline regimen, use of protease inhibitor in the baseline regimen, baseline hemoglobin (by steps of 0.5 g/dL), and platelet counts (by steps of 50 × 103/mm3) counts, history of HIV-associated thrombocytopenia (events reported by patients or clinicians), and treatment arm after randomization (IT or CT). A multivariate logistic regression analysis was then performed including in the models variables associated with the occurrence of thrombocytopenia in the univariate analysis with a P value < 0.10. Odds ratios (ORs) are given with 95% CIs.

To further understand the mechanisms of thrombocytopenia among the patients enrolled in this trial, we described the median platelet count in each arm during the study and assessed correlations between the changes from baseline in platelet count 8 weeks after the first treatment interruption in patients randomized in the IT arm, and those in CD4 and CD8 T-cell counts and in plasma HIV RNA levels using the Spearman rank correlation test.

Chi-square or Fisher exact tests were used to compare qualitative variables, and the Student test or the Wilcoxon rank-sum test was used to compare continuous variables.

Comparisons were made with use of a 2-sided alpha level of 0.05. Statistical analysis was performed with the use of SAS software version 9.1 (SAS Institute Inc, Cary, NC).

RESULTS

Twelve patients withdrew consent at baseline and 391 patients were included in the analysis, 197 in the IT arm, and 194 in the CT arm. Table 1 shows patients baseline characteristics which were well balanced across both treatment arms, with a median age of 42 years, 80% of men, 8% of patients with a previous AIDS-defining illness, a median nadir CD4 cell count of 280 cells/mm3, a median baseline CD4 count of 741 cells/mm3. Median duration of antiretroviral therapy before randomization was 5.2 years. Antiretroviral regimens consisted in a combination of nucleoside analogues [with 81% receiving lamivudine (3TC), 45% azidothymidine, 43% stavudine (d4T), 32% ddI] and either efavirenz (43%) or protease inhibitors (46%, boosted by ritonavir in 44%). Twelve (5 in IT arm, 7 in CT arm) had a history of HIV-related thrombocytopenia. During the study period, only 11 patients were lost to follow-up, 8 patients withdrew consent, 8 were censored after they reached immunological failure, and 2 died (1 sudden death of unknown origin and 1 hepatic failure with alcoholic cirrhosis). Overall, 92% of patients (n = 182) in the IT arm and 93% of patients in the CT arm (n = 180) completed the 96 weeks of the trial. Also, 43 patients (11%) discontinued their treatment strategy during the study, 27 (14%) in the IT arm, and 16 (8%) in the CT arm (P = 0.09 by the Log-rank test).

Incidence of Thrombocytopenia

Overall, 50 patients in the IT group (25.4%) and 19 (9.8%) in the CT group (P < 0.0001), developed thrombocytopenia (platelet count below 150 × 103/mm3) during the study (Fig. 1). Median time to thrombocytopenia was only 9 weeks in the IT arm [interquartile range (IQR): 8-40] as compared with 40 weeks (IQR: 9-74) in the CT arm. Figure 1 shows the Kaplan-Meier estimates of the occurrence of thrombocytopenia through 96 in the IT and CT arm (P < 0.0001, Log-rank test). Similar results were obtained in the on treatment analysis (data not shown).

Severe (grades 3-4) thrombocytopenia (platelet count below 50 × 103/mm3) occurred more frequently in the IT arm than in the CT: 4.6% (9 patients) versus 1% (2 patients), respectively (P = 0.03). Eighteen episodes of severe thrombocytopenia were observed in these 11 patients, which occurred in all but 2 cases after discontinuation of antiretroviral therapy (1 in CT arm and 1 in IT arm during a scheduled treatment period). Among the 11 patients with severe thrombocytopenia, 3 (2 IT and 1 CT) had a history of HIV-related thrombocytopenia and median baseline platelet count was 242 × 103/mm3 [211-280]. Four of these patients, all in the IT arm switched to the CT arm because of severe thrombocytopenia. Three patients, all in IT arm, had grade 4 thrombocytopenia (platelet count below 20 × 103/mm3) with hemorrhagic symptoms in 2 (bruising at venous puncture site and gynecological bleeding).

Risk Factors for Thrombocytopenia

Among all baseline variables tested in the univariate analysis, treatment arm (IT versus CT) with an OR of 3.13 (95% CI: 1.77-5.55), platelet count with an OR of 3.20 (95% CI: 2.24-4.58), baseline CD4 cell count with an OR of 1.25 (95% CI: 1.06-1.46), and a history of HIV-related thrombocytopenia with an OR of 7.16 (95% CI: 2.20-23.28), were all significantly associated with the occurrence of thrombocytopenia (Table 2). In the multivariate analysis, only treatment arm (IT versus CT) with an OR of 4.10 (95% CI: 2.14-7.85), platelet count with an OR of 3.41 (95% CI: 2.30-5.06), and a history of HIV-related thrombocytopenia with an OR of 11.87 (95% CI: 2.43-57.93) remained significantly associated with the occurrence of thrombocytopenia.

Figure 2 shows the median platelet count in both groups during the study (Figs. 2 and 3). At week 8, after the first treatment interruption, the median platelet count was 35 × 103/mm3 lower than baseline in the IT arm compared with 1 × 103/mm3 higher in the CT arm (P < 0.0001). Whereas the median platelet count remained almost unchanged in the CT arm, it decreased in the IT arm during each treatment interruption, and increased during periods on treatment. At week 96, the median platelet count was 16 × 103/mm3 lower than baseline in the IT arm compared with 12 × 103/mm3 lower in the CT arm (P = 0.01).

These changes in median platelet counts in the IT arms were similar to those previously reported with CD4 T-cell counts, and mirrored those of plasma HIV RNA levels and CD8 T-cell counts which increased during treatment interruptions and decreased under treatment.2

We therefore wished to assess correlations between changes from baseline to week 8 in platelet counts with those of CD4 T-cells, CD8 T-cells, and plasma viral load among patients randomized in the IT arm. As shown in Figure 3A, there was a moderate but significant correlation between changes in platelet counts and changes in CD4 T-cell counts (P < 0.0001), and an inverse correlation between platelet counts and plasma HIV RNA levels (P = 0.0005; Fig. 3B), whereas no correlation was found with changes in CD8 T-cells count (Fig. 3C).

DISCUSSION

Many clinical trials have addressed the issue of treatment interruption among patients with controlled HIV infection under antiretroviral therapy.1-4 Very few, however, have reported on the risk of thrombocytopenia associated with treatment discontinuation, although thrombocytopenia could potentially lead to severe clinical manifestations. In the Staccato trial, the authors reported a low incidence of thrombocytopenia, defined as a platelet count below 100 × 103/mm3, among patients randomized in the interruption arm (2.5%), which was not significantly higher than in the continuous arm (0%, P = 0.1).1 The same authors have also described 3 patients with recurrent thrombocytopenia in this same trial during periods off treatment.13 Maggiolo et al10 have reported the case of a patient with a history of HIV-related thrombocytopenia who experienced a platelet count below 1000/mm3, 4 months after treatment discontinuation, and who recovered after resuming antiretroviral therapy.

Using data from the Window trial, we were able to show that the risk of thrombocytopenia is high among patients interrupting their antiretroviral therapy, with a 96-week incidence of near 25% (Fig. 1). This incidence was significantly higher than among patients maintaining their antiretroviral therapy (9.8%, P < 0.0001 by the Log-rank test). Interestingly, median time to thrombocytopenia was only 9 weeks in the IT arm, and the main difference between arms was seen already at week 8, at the end of the first treatment interruption, with Kaplan-Meier plots remaining almost parallel between arms after week 8 (Fig. 1).

According to the definition of thrombocytopenia used in this study, a platelet count below 150 × 103/mm3, the clinical relevance of these observations could seem limited. However, 11 patients developed a severe thrombocytopenia with a platelet count below 50 × 103/mm3 during the study, after discontinuation of antiretroviral therapy in all but 2. Two of the patients were symptomatic with hematomas and gynecological bleeding. The consequences of these low platelet counts could have been more severe if antiretroviral therapy was not resumed after 8 weeks, and if platelet counts were not monitored every 8 weeks in this clinical trial. Fatal bleeding due to intracranial hemorrhage has been reported in patients with idiopathic thrombocytopenic purpura (ITP).14 It is, therefore, important to remind patients and physicians that even a short treatment interruption of a few weeks could lead to severe thrombocytopenia and that platelet counts should be carefully monitored if antiretroviral therapy has to be discontinued.

We then tried to identify baseline predictors of thrombocytopenia in this study. Among all baseline variables tested in our multivariate model (Table 2), being randomized in the treatment interruption arm, having a low baseline platelet count, and a history of HIV-related thrombocytopenia were all significantly associated with the occurrence of thrombocytopenia.

Other variables including CD4 cell count, either at baseline or at the nadir, were not associated with thrombocytopenia. Female sex was not found either as a risk factor for thrombocytopenia in this study. Also, the use of zidovudine or ddI in the antiretroviral regimen was not found to be associated with thrombocytopenia, probably because the reported beneficial effects of these drugs in patients with HIV-related thrombocytopenia are not specific, but rather the consequence of their antiviral activity.15-18 The benefit of antiretroviral therapy on HIV-related thrombocytopenia has indeed been well documented.16-20

Analyzing the changes in median platelet counts among patients randomized in the interruption arm during the 96 weeks of the Window trial, could also give insights into the pathogenesis of thrombocytopenia during HIV infection. Thrombocytopenia is indeed a common complication of HIV infection, and HIV-related thrombocytopenia resembles ITP, with normal or increased numbers of megakaryocytes in the bone marrow, and increased platelet-associated IgG autoantibodies. HIV-related thrombocytopenia is supposed to be the consequence of both a depressed platelet production secondary to HIV infection of megakaryocytes,15,21-26 and a reduction in platelet survival due to their increased peripheral destruction by macrophages through platelet-associated autoantibodies or circulating immune complexes, with sequestration in the spleen and sometimes the liver.15,22,27-30 Molecular mimicry between HIV antigens, mainly the envelope glycoprotein 120/160,31-34 and membrane-associated platelet glycoproteins such as IIB/IIIa, explains the cross-reactivity of these platelet-associated autoantibodies with HIV antigens.33,35,36 In patients with ITP, antibodies against glycoprotein IIb/IIIa probably originate from a limited number of B-cell clones, and these patients have increased numbers of HLA DR+ T-cells, and a cytokine profile suggesting T-cell activation, similar to what is found during HIV infection. The factors that initiate autoantibody production are unknown in patients with ITP, but it is tempting to hypothesize that HIV replication triggers the production of platelet autoantibodies in patients with HIV infection.37

During this study, median platelet count indeed decreased during each treatment interruption and increased to near the baseline value after each period on treatment (Fig. 2). These changes in platelet counts were fast, and followed the same course previously reported with CD4 cell counts in this study. Also, changes in platelet counts mirrored those seen with plasma HIV RNA levels during periods on and off treatment. Interestingly, week 8 changes from baseline in median platelet counts were significantly positively correlated with changes in CD4 T-cells, and negatively correlated with plasma HIV RNA levels. Such an association between platelet counts and plasma HIV RNA levels has already been reported in cohorts of HIV-infected hemophiliacs.38 The decrease of platelet counts observed during each treatment interruption is therefore likely to be the direct consequence of HIV replication.

In conclusion, this study demonstrated that antiretroviral therapy interruption among patients with well-controlled HIV infection, could lead to the rapid occurrence of thrombocytopenia, especially among those with a low baseline platelet count or a history of HIV-related thrombocytopenia. Treatment interruption should therefore be strictly forbidden for such patients. The decrease in platelet count during treatment interruption is correlated to the decrease in CD4 T-cells, and negatively correlated to plasma HIV RNA levels. This risk of thrombocytopenia is therefore another limitation of IT, which should be avoided as much as possible.